Effect of a new dietary supplement formula on the activity and expression of pro-inflammatory genes in vitro

Abstract

Purpose : Wet Age-related Macular Degeneration and Diabetic Macular Edema are characterized by chronic inflammation and high levels of Vascular Endothelial Growth Factor (VEGF). Since anti-VEGF agents do not possess a direct effect on inflammation, we set out to investigate the potential anti-inflammatory activity of a nutritional supplement formula codenamed AVS (SIFI S.p.A.) as a candidate support to anti-VEGF therapies. To this end, we exposed stimulated J774.2 and A549 cells to AVS in order to evaluate the effect exerted upon expression and/or activity of interleukine-1beta (IL-1β) and inducible nitric oxide synthase (iNOS) or cyclooxygenase-2 (COX-2), respectively.

Methods : J774.2 and A549 cells grown to sub-confluence were pre-treated (2 h) with AVS (1.9 mg/ml) and then stimulated with LPS (1 µg/ml) or IL-1β (10 ng/ml), respectively. Following overnight incubation, the medium was collected and nitrites or prostaglandin E₂ (PGE₂) production were determined. Total RNA was also extracted to assess the expression of IL-1β, iNOS and COX-2 mRNAs by Real Time RT-PCR. Data represent the average of 8 replicates from 4 different experiments. Statistically relevant differences were sought by unpaired t-test.

Results : LPS treatment induced the accumulation of ~70 µmol/l nitrites in the J774.2 cell culture medium. Treatment with AVS inhibited the accumulation of nitrites by 89% compared to control (p≤0.001). Consistently, AVS produced a significant (p≤0.0001) inhibition of iNOS and IL-1β mRNA expression by 670-fold (99%) and 1100-fold (96%), respectively. COX-2 mRNA expression was not affected by AVS treatment in IL-1β-stimulated A549 cells. However, AVS effectively inhibited PGE₂ accumulation by 99% compared to control (p≤0.001).

Conclusions : The specific composition of AVS was shown to be endowed with anti-inflammatory properties. Indeed, AVS was effective in inhibiting nitrites accumulation in J774.2 cultures, most likely by inhibiting the expression of iNOS and IL-1β genes. Interestingly, AVS effectively inhibited the synthesis of PGE₂ in A549 cells while unable to inhibit COX-2 expression. These findings suggest that AVS is able to act at multiple levels modulating the expression and/or the activity of important pro-inflammatory genes. Therefore, the AVS formula may well prove a useful anti-inflammatory aid in patients undergoing standard anti-VEGF therapy.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.